The invention relates to signal transduction inhibitors.
The c-Jun NH2-terminal kinase (JNK) is a member of the stress-activated group of Mitogen Activated Protein kinases (MAP kinases) implicated in the control of cell growth. The JNK signal transduction pathway is activated in response to environmental stress and by the engagement of several classes of cell surface receptors, including cytokine receptors, serpentine receptors, and receptor tyrosine kinases. Whitmarsh et al., J. Mol. Med., 74:589 (1996). In addition, genetic studies of Drosophila have demonstrated that JNK is required for early embryonic development. Sluss et al., Genes and Dev., 10:2745 (1996); Riesgo-Escovar et al., Genes and Dev. 10:2759 (1996). In mammalian cells, JNK has been implicated in the immune response, oncogenic transformation, and apoptosis. JNK mediates these-effects, at least in part, by increasing the expression of target genes. Targets of the JNK signal transduction pathway include the transcription factors c-Jun, ATF2, and Elk-1. Whitmarsh et al., supra.
JNK is activated in the liver by metabolic oxidative stress. Mendelson et al., Proc. Natl. Acad. Sci. USA 93:12908-12913 (1996). Activation of JNK also occurs in the kidney during stress, for example, during Bchemic renal failure. Demari et al., Am. J. Physiol. 272:F292-F298 (1997). JNK is also activated during cardiovascular disease such as ischemia Ireperfusion and during organ transplantation. Pombo et al., J. Biol. Chem. 269:26546-26551 (1994); Force et al., Circ. Res. 78:947-53 (1996).
While JNK is located in both the cytoplasmic and the nuclear compartments of quiescent cells, activation of the JNK signal transduction pathway is associated with accumulation of JNK in the nucleus. Mechanisms governing this sub-cellular distribution have not been previously elucidated.
Anchor or tethering proteins play an important role in the regulation of multiple signal transduction pathways. These anchor proteins, which include the nuclear factor kappa B (NFkB) inhibitor IkB, the A kinase anchor protein (AKAP) group of proteins that bind the type II cyclic adenosine monophosphate (AMP) dependent protein kinase, and the p190 protein that binds Ca2+-calmodulin-dependent protein kinase II, localize their tethered partners to specific sub-cellular compartments. Verma et al., Genes and Dev., 9:2723 (1995); McNeill et al., J. Biol. Chem., 270:10043 (1995); Faux et al., Trends Biochem. Sci., 21:312 (1996)). Anchor proteins also target enzymes to specific substrates, and create multi-enzyme signaling complexes, such as the Ste5 MAP kinase scaffold complex and the AKAP79 kinase/phosphatase scaffold complex. Choi et al., Cell, 78:499 (1994); Klauck et al., Science, 271:1589 (1996); Faux et al., Cell, 85:9 (1996)).
The invention, which is based on the discovery of a cytoplasmic anchor protein, JNK-interacting protein 1 (JIP-1; SEQ ID NO:1), features JIP-1 polypeptides and nucleic acids, therapeutic compositions containing these polypeptides and nucleic acids, and methods of administering these compositions. JIP-1 specifically binds to and inhibits the biological effects of JNK, including the initiation of apoptosis and oncogenic transformation. JIP-1 is therefore useful as a therapeutic agent for treating pathological conditions characterized by apoptosis or transformation. For example, JIP-1 compositions can be used to treat neurodegenerative diseases characterized by apoptosis, including Parkinson""s disease and Alzheimer""s disease; and blood clots, which left untreated could result in stroke and associated memory loss. Other conditions that can be treated using the compositions and methods of the invention are autoimmune diseases such as arthritis; other conditions characterized by inflammation; and malignancies, such as leukemias, e.g., chronic myelogenous leukemia (CML). other conditions that can be treated with JIP-1 compositions include oxidative damage to organs such as the liver and kidney, and heart disease, particularly damage due to ischemia/reperfusion and cardiomyopathy. JIP-1 compositions can also be used to treat donor organs for transplantation. These organs are exposed to substantial environmental stress, the effects of which are blocked by JNK inhibitors such as JIP-1.
The invention features a substantially pure JIP-1 polypeptide. A xe2x80x9cJIP-1 polypeptidexe2x80x9d is a protein having an amino acid sequence that specifically binds JNK to the same extent, or at least 10% of the binding activity of wildtype JIP-1. Such polypeptides can be from 5 to 200 amino acids in length, e.g., from 10 to 100 amino acids in length, or from 20 to 50 amino acids in length. Such polypeptides include the JNK Binding Domain (JBD), or portions thereof, of JIP-1 (e.g., amino acids 148 to 174, forming the xe2x80x9ccorexe2x80x9d of the JBD of wildtype JIP-1, shown in FIG. 2, and having the sequence SGDTYRPKRPTTLNLFPQVPRSQDTLN; SEQ ID NO:3). JIP-1 polypeptides are preferably derived from a mammal, such as a mouse or a human.
In various embodiments, the polypeptide is soluble, the polypeptide includes the JNK-binding domain of JIP-1 or a portion thereof, the polypeptide is at least 80%, 90%, or 100% identical to the amino acid sequence from amino acid 148 to amino acid 174 of JIP-1 (the core JNK-binding domain; SEQ ID NO:3), or the polypeptide has an amino acid sequence identical to the amino acid sequence from amino acid 148 to amino acid 174 of JIP-1 (SEQ ID NO:3), or the polypeptide is at least 80%, 90%, or 100% identical to the amino acid sequence from amino acid 127 to amino acid 281 of JIP-1 (the JNK-binding domain; SEQ ID NO:4).
The polypeptides of the invention can be modified to enhance their uptake by cells. Such modifications increase the hydrophobicity of molecules to facilitate passage through the lipid bilayer of the cell membrane. For example, polypeptides can be complexed with myristic acid or packaged in liposomes. Alternatively, JIP-1 polypeptides can be complexed with hydrophobic moieties (e.g., lipids) or peptides that increase the delivery of proteins into cells.
The invention also includes peptide mimetics of JIP-1 polypeptides. A xe2x80x9cpeptide mimeticxe2x80x9d of a known polypeptide is a compound that mimics the activity of the peptide or polypeptide, but which is composed of molecules other than, or in addition to, amino acids.
By xe2x80x9cpolypeptidexe2x80x9d is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation), and thus includes peptides, proteins, and fusion proteins.
A xe2x80x9csubstantially identicalxe2x80x9d polypeptide sequence differs from a given sequence only by conservative amino acid substitutions or by one or more nonconservative substitutions, deletions, or insertions located at positions which do not destroy the function of the polypeptide compared to wildtype JIP-1. Polypeptides of the invention can be 70%, 80%, 85%, 90%, or 95% identical to wildtype JIP-1.
A xe2x80x9csubstantially purexe2x80x9d preparation is at least 60% by weight of the compound of interest, e.g., a JIP-1 polypeptide or fragment of a JIP-1 polypeptide. Preferably the preparation is at least 75%, more preferably at least 90%, and more preferably at least 95% by weight of the compound of interest. Purity can be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or High Pressure Liquid Chromatography (HPLC) analysis.
The polypeptides of the invention include, but are not limited to, recombinant polypeptides, natural polypeptides, and synthetic polypeptides, as well as preproteins or proproteins and biologically active fragments. A xe2x80x9cbiologically active fragmentxe2x80x9d of JIP-1 is a fragment having at least 50%, 70%, 80%, 90%, 95%, or 100% or greater, of the activity of naturally occurring or synthetic, full length JIP-1.
The polypeptides of the invention can be physically linked to another polypeptide, e.g., a marker polypeptide. For example, the polypeptide can be fused to a hexa-histidine tag to facilitate purification of bacterially expressed proteins, or a hemagglutinin tag to facilitate purification of protein expressed in eukaryotic cells.
In another aspect, the invention features an isolated nucleic acid that includes a sequence encoding a JIP-1 polypeptide or a fragment of such a polypeptide. Preferably, the nucleic acid is derived from a mammal.
The invention also encompasses nucleic acids that hybridize under stringent conditions (as described herein) to a nucleic acid encoding a JIP-1 polypeptide. Stringent conditions include hybridization at 68xc2x0 C. in 5xc3x97SSC/5xc3x97Denhardt""s solution/1.0% SDS, or in 0.5 M NaHPO4 (pH 7.2)/1 mM EDTA/7% SDS, or in 50% formamide/0.25 M NaHPO4 (pH 7.2)/0.25 M NaCl/1 mM EDTA/7% SDS; and washing in 0.2xc3x97SSC/0.1% SDS at room temperature or at 42xc2x0 C., or in 0.1xc3x97SSC/0.1% SDS at 68xc2x0 C., or in 40 mM NaHPO4 (pH 7.2)/1 mM EDTA/5% SDS at 50xc2x0 C., or in 40 mM NaHPO4 (pH 7.2) 1 mM EDTA/1% SDS at 50xc2x0 C. Moderately stringent conditions include washing in 3xc3x97SSC at 42xc2x0 C. The parameters of salt concentration and temperature can be varied to achieve the desired level of identity between the probe and the target nucleic acid. For guidance regarding such conditions see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.; and Ausubel et al., supra, at Unit 2.10.
The hybridizing portion of the hybridizing nucleic acid is preferably 20, 30, 50, or 70 bases long. The hybridizing portion of the hybridizing nucleic acid can be 95% or even 98% or 100% identical to the sequence of a portion of a nucleic acid encoding a JIP-1 polypeptide. Hybridizing nucleic acids of the type described above can be used as a cloning probe, a primer (e.g., a PCR primer), or a diagnostic probe. Preferred hybridizing nucleic acids encode a polypeptide having some or all of the biological activities possessed by naturally-occurring JIP-1. Thus, they may encode a protein that is shorter or longer than the various forms of JIP-1 described herein. Hybridizing nucleic acids can also encode proteins that are related to JIP-1, e.g., proteins encoded by genes that include a portion having a relatively high degree of identity to a JIP-1 gene described herein.
The term xe2x80x9cnucleic acidxe2x80x9d encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. The nucleic acid may be double-stranded or single-stranded. Where single-stranded, the nucleic acid may be the sense strand or the antisense strand.
An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid that is free of the nucleic acids that normally flank it in the genome. The term therefore includes, e.g., a recombinant nucleic acid incorporated into a vector, such as an autonomously replicating plasmid or virus; a cDNA or genomic DNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease treatment; and recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequences.
A xe2x80x9csubstantially identicalxe2x80x9d nucleic acid is a nucleic acid with a sequence that is at least 50%, preferably 70%, and more preferably 85%, 90%, or 95% homologous to a given nucleic acid sequence, e.g., SEQ ID NO:2.
The invention also features transformed cells harboring a nucleic acid encompassed by the invention. Vectors and plasmids that include a nucleic acid properly positioned for expression are also within the invention. A xe2x80x9ctransformed cellxe2x80x9d is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding a JIP-1 polypeptide.
xe2x80x9cOperably linkedxe2x80x9d means that the selected DNA molecule is positioned adjacent to one or more sequence elements that direct transcription and/or translation of the sequence such that the sequence elements can control transcription and/or translation of the selected DNA (i.e., the selected DNA is operably associated with the sequence elements). Such operably associated elements can be used to facilitate the production of a JIP-1 polypeptide.
The invention also features purified antibodies which specifically bind a JIP-1 protein or polypeptide. A xe2x80x9cpurified antibodyxe2x80x9d is an antibody which is at least 60%, by dry weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. The preparation can be at least 75%, at least 90%, and up to 99% or more, by dry weight, antibody.
An antibody that xe2x80x9cspecifically bindsxe2x80x9d an antigen recognizes and binds to that antigen, e.g., a JIP-1 polypeptide.
Also within the invention are antisense molecules and ribozymes for inhibiting JIP-1 expression.
The invention also features antagonists and agonists of JIP-1. Antagonists can inhibit one or more of the functions of JIP-1. Suitable antagonists can include large or small molecules, antibodies to JIP-1, and JIP-1 polypeptides that compete with a native form of JIP-1. Such antagonists include SEQ ID NO:3, a component of an active site of JIP-1, i.e., the JNK-binding domain. Agonists of JIP-1 will enhance or facilitate one or more of the functions of JIP-1. Agonists and Antagonists include polyproline motifs, which bind to SH3 domains such as that found in JIP-1.
A xe2x80x9ctherapeutically effective amountxe2x80x9d of a substance is an amount capable of producing a medically desirable result in a treated patient, e.g., inhibition of the expression or activity of a specific protein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the arts of protein chemistry or molecular biology. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described infra. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from the detailed description and the claims.